Abstract

Background Exenatide is a glucagon-like peptide (GLP)-1 analogue with insulinotropic and insulinomimetic properties. Because insulin and GLP-1 have been described as reducing apoptosis, exenatide might confer cardioprotection after acute myocardial infarction (MI).

Methods Pigs were randomized to exenatide or phosphate-buffered saline (PBS) treatment after 75 min of coronary artery ligation and subsequent reperfusion. Infarct size was assessed with Evans Blue (Sigma-Aldrich, St. Louis, Missouri) and triphenyltetrazolium chloride. Cardiac function was measured with epicardial ultrasound and conductance catheter-based pressure-volume loops. Western blotting, histology, and activity assays were performed to determine markers of apoptosis/survival and oxidative stress.

Myocardial infarction (MI) results in irreversible loss of cardiomyocytes. Restoration of the antegrade coronary flow in the infarct-related coronary artery limits myocardial ischemic necrosis and is the cornerstone treatment of ST-segment elevation myocardial infarction (STEMI). However, despite adequate reperfusion, most patients still suffer irreversible myocardial cell loss, which is partly caused by the reperfusion itself (1). Reperfusion induces several abrupt biochemical and metabolic changes, including the generation of reactive oxygen species leading to oxidative stress, intracellular calcium overload, the rapid restoration of physiologic pH, and inflammation (2). These changes eventually interact to accelerate myocardial apoptosis through the opening of the mitochondrial permeability transition pore. To prevent excessive loss of myocardial tissue in patients undergoing early reperfusion therapy, the development of novel therapeutics to reduce or prevent reperfusion injury is of major importance.

Glucose-insulin-potassium infusion has been postulated to possess cytoprotective potential and has been investigated in several clinical trials for cardioprotection in patients with STEMI. Its efficacy is controversial, with some studies reporting a reduction in mortality in patients with acute MI (3–5) and most studies demonstrating no beneficial effect (6–8). A better alternative might be exenatide (exendin-4), a 39 amino acid peptide originally derived from the saliva of the gila monster, a venomous lizard native to the southwestern U.S. and northern Mexico. It mimics human glucagon-like peptide (GLP)-1, a gut incretin hormone that is released by the gut in response to nutrient intake (9). It exerts insulinotropic and insulinomimetic properties via the G-protein–coupled GLP-1 receptor, which has also been reported to be expressed in the heart (10,11). The GLP-1 has been shown to be cardioprotective in a rat model of myocardial ischemia and reperfusion (I/R) injury (12).

The purpose of this study is to investigate whether exenatide is capable of reducing myocardial infarct size. To establish this, we use a clinically relevant large animal model of myocardial I/R injury. We hypothesize that exenatide confers cardioprotection in this model by reducing myocardial apoptosis, resulting in reduced myocardial infarct size and improved cardiac function.

Methods

Animals

All experiments were performed in accordance with the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources and with prior approval by the Animal Experimentation Committee of the Faculty of Medicine, Utrecht University, the Netherlands.

Study design

Eighteen Dalland Landrace pigs (70 to 80 kg) were pretreated with clopidogrel 75 mg for 3 days and amiodarone 400 mg for 10 days to prevent thrombotic complications and arrhythmias, and the treatment was continued after the surgery. Myocardial I/R injury was induced surgically, under general anesthesia as described previously (13), by left circumflex coronary artery (LCx) occlusion for 75 min and subsequent reperfusion for 3 days. Before ischemia, the animals received 300-mg amiodarone for 30 min, followed by 100 mg/h during the surgical procedure, and 100 IU/kg body weight heparin intravenously. Animals were randomly assigned to exenatide treatment (n = 9, 10 μg SC and 10 μg IV 5 min before the onset of reperfusion and 10 μg twice daily SC—a normal human dose—on the following 2 days) or placebo phosphate-buffered saline (PBS) treatment (n = 9) following the same treatment regimen. The treatment was initiated just before reperfusion to ensure a systemic concentration at the beginning of reperfusion. All animals were killed after 3 days of reperfusion. Additional pigs (PBS n = 4, exenatide n = 4) underwent a sham operational procedure during which the suture around the coronary artery was removed without closing it. Furthermore, additional pigs (PBS n = 4, exenatide n = 4) were included for harvesting myocardial tissue 2 h and 4 h after reperfusion for the assessment of molecular pathways involved in myocardial protection.

I/R injury and operational procedure

During the operation, electrocardiogram, arterial pressure, and capnogram were continuously monitored. After median sternotomy, a pressure-tipped Millar catheter was inserted through the apex into the left ventricle (LV) to measure LV pressure. Cardiac output was measured with a transonic flow probe (Transonic Systems Inc., Ithaca, New York), which was placed around the proximal aorta. Ischemia was induced by temporary proximal LCx ligation with a 2-0 prolene suture. Reperfusion was established by suture release after 75 min of ischemia.

Pressure-volume loops

Pressure-volume loops were assessed with a conductance catheter as described previously (13). In short, LV pressure and volume signals derived from the conductance catheter were displayed and acquired with a Leycom CFL-512 (CD Leycom, Zoetermeer, the Netherlands). Data were acquired during steady state and during temporal caval vein occlusion, while ventilation was paused at end-expiration. Analysis of the pressure-volume loops was performed with custom software as previously described (14). Systolic function was characterized by LV ejection fraction, dP/dtMAX, and ES elastance, which was determined by the slope of the ES pressure-volume relationship. Diastolic chamber stiffness was quantified by linear regression of the ED pressure-volume relationship (15).

Infarct size

Infarct size was assessed with Evans Blue (Sigma-Aldrich, St. Louis, Missouri) and triphenyltetrazolium chloride staining as described previously (16). In short, after 3 days of reperfusion, the proximal LCx was re-occluded at exactly the same location, and Evans Blue (Sigma-Aldrich) (1%) was infused into the remaining coronary arteries, allowing determination of the area at risk (AAR). After excision of the heart, the LV was cut into 5 slices from apex to base and incubated in 1% triphenyltrazolium chloride (Sigma-Aldrich Chemicals, Zwijndrecht, the Netherlands) in 37°C Sörensen buffer (13.6 g/l potassium dihydrogen phosphate + 14.2 g/l disodium hydrogen phosphate, pH 7.4) for 15 min to discriminate infarct tissue from viable myocardium.

Insulin and glucose levels

Serum insulin was analyzed on an Immulite 1000 immunochemistry system (Siemens Medical Solutions Diagnostics, Tarrytown, New York), and plasma glucose was analyzed on a DxC 800 routine chemistry system (Beckman Coulter, Brea, California).

Immunostaining

Tissue samples from the border region and remote region were fixated in 4% formalin before being embedded in paraffin. The border zone was chosen as the semi-injured region within 4 mm from the infarct zone. Samples from the LV septum were chosen as remote area. After antigen retrieval in 10-mmol/l citric acid, the tissue sections were incubated with 10% normal horse serum for 30 min, mouse–anti-8-hydroxydeoxyguanosine (OHdG) (OXIS international, Foster City, California) 1:20 in PBS containing 0.1% (wt/vol) bovine serum albumin overnight at 4°C, biotin labeled horse–anti-mouse (Vector Laboratories, Burlingame, California) 1:500 for 1 h, and with streptavidin-horseradish peroxidase 1:1,000 for 1 h. Finally, the sections were incubated with hydrogen peroxide-diaminobenzidine for 10 min. To assess myocardial apoptosis, a terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) was performed according to the manufacturer's instructions (Roche, Woerden, the Netherlands), and eventually the deoxyribonucleic acid nick ends were visualized with diaminobenzidine. The sections were then shortly counterstained with hematoxylin and eosin to visualize the cells. The amount of 8-OHdG–positive nuclei and the amount of TUNEL-positive nuclei was quantified in 4 randomly picked fields/section with digital image microscopy software (Analysis, Olympus, Münster, Germany) at 200× magnification and averaged. The amount of TUNEL-positive cells was expressed as a percentage of the total amount of cells.

Oxidative stress assay

Superoxide dismutase activity and catalase activity were measured with activity assay kits according to the manufacturers' protocols (Calbiochem [San Diego, California] and Cayman Chemical [Ann Arbor, Michigan], respectively). For both assays, 10-μl protein samples were loaded into the wells, and the values were corrected for the protein concentration in the sample afterwards.

Data analysis

All analyses were performed in a blinded fashion. Statistical analysis was performed with SPSS version 15.0 (SPSS Inc., Chicago, Illinois). Values are presented as mean ± SEM. Statistical comparison of infarct size, pressure-volume loop derived functional parameters, histology, and protein expression levels was performed with the Student t test. Hemodynamic and echocardiographic functional parameters, insulin, and glucose levels were compared with a 2-way analysis of variance for repeated measurements with Bonferroni post hoc tests. Values of p < 0.05 were considered significant.

Myocardial infarct size quantification as a percentage of the area at risk (AAR) (A) and as a percentage of the total left ventricle (B). Phosphate-buffered saline (PBS) n = 9; exenatide n = 9. Representative pictures after Evans Blue (Sigma-Aldrich) and triphenyltetrazolium chloride staining are shown in C and D. Blue indicates non-threatened myocardium, red indicates the noninfarcted area within the area at risk, and white indicates myocardial infarction.

Cardiac function

Both local and global cardiac function were decreased 1 h after I/R injury. Three days later, however, recovery of systolic wall thickening and fractional area shortening was increased in animals treated with exenatide compared with PBS-treated animals (Figs. 2A and 2B). Beta1-adrenergic stimulation with dobutamine increased functional improvement to a greater extent in animals treated with exenatide compared with animals treated with PBS. The ED and ES volumes were lower in exenatide-treated pigs compared with control pigs, indicating that exenatide prevents acute LV dilation after MI and maintains contractile performance (Figs. 3A and 3B). Also, other global functional parameters, such as LV ejection fraction (Fig. 3C), dP/dtMAX (Fig. 3D, Table 1), and ES elastance (Fig. 3E) were higher after exenatide treatment. In addition, myocardial stiffness was lower (Fig. 3F), indicating that diastolic function also improved. Furthermore, the ED pressure increased significantly in PBS-treated animals but not in animals treated with exenatide (Table 1). Heart rates increased equally in both groups (Table 1). Exenatide did not influence cardiac function in sham-operated animals (Fig. 3, Table 1).

Insulin and glucose levels

In pigs treated with exenatide, the increase in serum insulin levels 4 h after reperfusion was significantly more pronounced compared with control animals, confirming the insulinotropic properties of exenatide in pigs (Fig. 4A). Plasma glucose levels, however, were similar at all times in both groups, suggesting that exenatide treatment entails a low risk of hypoglycemia (Fig. 4B). Hypoglycemia (plasma glucose level <3.3 mmol/l or <60 mg/dl) occurred in 1 pig (11%) treated with PBS 4 h after reperfusion and in none of the pigs treated with exenatide.

Apoptosis and oxidative stress

Different markers of apoptosis and survival were assessed at different time points after reperfusion to elucidate potential protection mechanisms by exenatide. Two hours after reperfusion, expression of phosphorylated Akt (pAkt) was increased in myocardial tissue of animals that were treated with exenatide compared with PBS-treated animals (Fig. 5A). Four hours after reperfusion, levels of active caspase 3, an effector caspase that induces apoptosis, were lower in treated animals compared with control animals (Fig. 5B). Also, expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax were determined. Whereas no differences were observed between treated and control animals 2 h and 4 h after reperfusion, Bcl-2 expression levels and Bcl-2/Bax ratios were higher in treated animals 72 h after reperfusion (Fig. 5C). Activity of the antioxidant enzymes superoxide dismutase and catalase were increased in exenatide-treated animals 72 h after reperfusion. Nuclear oxidative stress and deoxyribonucleic acid fragmentation as assessed with 8-OHdG staining and TUNEL assay, respectively, were reduced in border areas of pigs treated with exenatide compared with control pigs (Fig. 6). TUNEL positive cells were rarely found in the remote area (0.12 ± 0.08% [PBS] vs. 0.10 ± 0.07% [exenatide]; p = 0.849) or in sham-operated animals. All molecular and histological data are summarized in Table 2.

Discussion

Cardioprotection has been extensively investigated in the past 30 years, since the beginning of the reperfusion era. Successful results have been obtained in numerous experimental studies. However, translation of experimentally promising results into clinical therapy has proven to be difficult (2,18). To date, no compound has proven clinical efficacy in reducing myocardial infarct size in combination with reperfusion therapy. Barriers on the experimental level might include the use of non-clinically applicable experimental models (e.g., isolated myocytes, genetically modified animals), improper timing of compound administration (before ischemia, during early ischemia), and the lack of emphasis on clinically relevant end points. To increase translational efficiency, it is mandatory to conduct appropriately designed pre-clinical studies in large animal models with assessment of clinically relevant end points, such as mortality, myocardial infarct size, and cardiac function. In this study, we investigated the cardioprotective properties of exenatide in a clinically compliant pig model of myocardial I/R injury. To induce a pathological substrate that is comparable to that of the average patients with acute MI, we selected a period of 75 min of ischemia, although this is shorter than the average time from onset of ischemia to reperfusion in patients. In pigs, 75 min of ischemia induces a myocardial infarct of 53.6 ± 3.9% of the AAR. Protective mechanisms such as collateralization, ischemic preconditioning, and residual coronary flow might account for delay in cardiac cell death during ischemia in patients. Pigs closely resemble humans with regard to cardiovascular anatomy and physiology, the treatment was given just before reperfusion, a reperfusion period of 3 days was allowed before assessment of infarct size, and clinically relevant end points were determined. In this model, exenatide reduced myocardial infarct size by 40% and significantly improved systolic and diastolic cardiac performance. This was associated with increased insulin serum levels without changes in plasma glucose levels, reduced nuclear oxidative stress, and reduced apoptosis.

Bose et al. (12) have previously shown that GLP-1 protects the rat heart from I/R injury. However, GLP-1 was infused during the entire ischemic period, rendering uncertainty as to whether GLP-1 reduced reperfusion injury or ischemic injury or both. This complicates clinical translation, because most patients are referred to the clinic after an ischemic period. In an isolated rat heart model, however, GLP-1 was demonstrated to also exert protective effects when administered during reperfusion (19). Clinical applicability of GLP-1 is complicated by its very short half-life of several minutes, due to rapid breakdown by dipeptidyl peptidase IV, which requires continuous infusion of GLP-1. Exenatide is approved by the Food and Drug Administration for glucose control in patients with type 2 diabetes type (20,21). It has a longer half-life (60 to 90 min) compared with GLP-1, due to resistance to degradation by dipeptidyl peptidase IV, and can be administered subcutaneously in small volumes (22). This combination of favorable properties makes exenatide more attractive for clinical application compared with GLP-1 and also compared with glucose-insulin-potassium. Importantly, the risk of hypoglycemia, which regularly causes problems with other antidiabetic agents, such as sulphonylureas, is reduced with exenatide (20). Also in our study, hypoglycemia did not develop in pigs treated with exenatide.

Several mechanisms might be responsible for the cardioprotective effects of exenatide. First, glucose metabolism is stimulated over fatty acid metabolism, which is more efficient with respect to oxygen consumption for adenosine triphosphate production (23), and which might therefore reduce oxygen demand. Second, exenatide might reduce myocardial apoptosis and oxidative stress. The GLP-1 can protect the rat heart from I/R injury, mediated by the prosurvival kinases PI3K/Akt, p42/44 PKA, and P70s6 (12,19). In the present study, myocardial expression of pAkt was increased after exenatide treatment and expression of active caspase 3 were reduced. Also, activity of the antioxidant enzymes superoxide dismutase and catalase were higher in animals treated with exenatide, and nuclear oxidative stress was reduced. Alternatively, increased serum levels of insulin, which can directly exert anti-apoptotic effects via the insulin receptor (24–26), might have conferred cytoprotection. The fact that GLP-1 protects against I/R injury in isolated hearts, however, suggests the presence of mechanisms independent of circulating insulin (19).

The salvage of myocardial tissue by exenatide was accompanied by increased cardiac systolic and diastolic performance. After Dobutamine infusion, systolic function increased to a greater extent in treated animals, which likely reflects a reduced number of irreversibly injured cardiomyocytes in these pigs.

Besides preservation of cardiomyocytes, other factors could have contributed to enhanced cardiac function. Oxidative stress is an important mediator of myocardial stunning, which refers to sustained functional impairment after ischemia of normally perfused, viable myocardium. Also, inhibition of caspase 3 has been demonstrated to reduce stunning (27). Stunning can prolong for days or even weeks after ischemia and can contribute to cardiac failure in the acute phase after MI. In a previous study, GLP-1 was demonstrated to reduce stunning in a dog model of brief coronary occlusion and reperfusion (28). We have demonstrated that oxidative stress and caspase 3 expression were reduced after exenatide treatment, which might have alleviated myocardial stunning. Finally, exenatide induces metabolic changes in the heart as fatty acid metabolism shifts to carbohydrate metabolism (23), which demands less oxygen for adenosine triphosphate production. The GLP-1 infusion improves myocardial contractile performance in a canine model of pacing-induced dilated cardiomyopathy (29). In a small clinical study with acute MI and successful reperfusion, GLP-1 infusion improved regional and global LV function (30). These beneficial effects contributed to improved metabolic efficiency, and data on myocardial infarct size were not available in these studies.

We have not been able to investigate the effect of common comorbidities such as hypertension, dyslipidemia, and diabetes, which might influence the impact of exenatide on cardioprotection. Cardioprotective effects of exenatide in diabetic patients might even be more pronounced, because hyperglycemia is an independent factor associated with a poor outcome, and glycemic control is associated with a better outcome in the setting of acute MI (31–33). The clinical efficacy of exenatide, however, remains to be investigated in the near future.

Conclusions

We provide compelling evidence that exenatide confers strong cardioprotection and improves LV systolic and diastolic function in a clinically relevant large animal model of I/R injury. These data identify exenatide as a promising compound for myocardial salvage in patients with STEMI in combination with reperfusion therapy.

Acknowledgments

Footnotes

This work was funded by the Netherlands Heart Foundation (NHS), grant 2005T022. This study was supported by the NHS (to. Dr. Timmers) and the Netherlands Organization for Scientific Research NWO (to Dr. Hoefer). Dr. de Kleijn is a cofounder of a small biotech, but there is no conflict of interest with the study. Dr. DeVries is an advisor and on the Speakers' Bureau for Liraglutide, a GLP-1 analog from Novo Nordisk. Dr. Pasterkamp is a cofounder of a start-up company, Cavadis; however, there is no conflict of interest related to this study.

(1993) Exendin-4 is a high potency agonist and truncated exendin-(9-39)-amide an antagonist at the glucagon-like peptide 1-(7-36)-amide receptor of insulin-secreting beta-cells. J Biol Chem268:19650–19655.

(2005) Glucagon like peptide-1 is protective against myocardial ischemia/reperfusion injury when given either as a preconditioning mimetic or at reperfusion in an isolated rat heart model. Cardiovasc Drugs Ther19:9–11.

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